There are a number of known methods useful in staining proteins which utilize silver. For example, L. Kerenyi, et al., Clin. Chim. Acta 38, 465-467 (1972) describes a method for demonstrating proteins in electrophoretic, immunoelectrophoretic and immunodiffusion preparations whereby the preparations are treated with potassium ferrocyanide which is transformed during development into silver ferrocyanide then into colloidal silver grains. The physical developer contains anhydrous sodium carbonate, ammonium nitrate, silver nitrate, tungstosilicic acid and formalin, and the protein in the preparations stain dark brown with a pale gray background.
R. C. Switzer, et al., Anal. Biochem. 98, 231-237 (1979) and C. R. Merril, et al., Proc. Nat'l. Acad. Sci. U.S.A., 76, No. 9, 4335-4339 (1979) describe a silver stain technique for detecting proteins and peptides in polyacrylamide gel which is a modification of de Olmos' neural, cupric-silver stain. The procedure consists of ten steps and utilizes an aqueous solution of silver nitrate and cupric nitrate and involves treatment with a diammine solution which is known to sometimes form an explosive silver amide complex. The proteins stain as dark spots on a darkened background.
B. A. Oakley, et al., Anal. Biochem. 105, 361-363 (1980) simplified the above procedure of Switzer, et al., by reducing the number of steps involved to six and also reducing the amount of silver required without diminishing the sensitivity of the technique. However, the manner in which the proteins stain was not changed, i.e., dark stain on a darkened background.
A further modification of the Switzer, et al., procedure was made by R. C. Allen, Electrophoresis I, 32-37 (1980) who increased the sodium to ammonium ion ratio which resulted in increased silver deposition.
C. R. Merrill, et al., Anal. Biochem. 110, 201-207 (1981) modified and simplified the above procedure of Kerenyi, et al., adapting it to acrylamide gels.
D. Goldman, et al., Clin. Chem. 26, No. 9, 1317-1322 (1980) report that when using a procedure essentially the same as that of Merrill, et al., (PNAS, 1976) and Switzer, et al., (Anal. Biochem., 1979) proteins from samples of cerebral spinal fluid stained in shades of yellow, red and blue.
C. R. Merrill, et al., Science 211, 1437-1438 (1981) describe a silver stain procedure for proteins separated by two dimensional gel electrophoresis which requires treatment with potassium dichromate and nitric acid prior to staining with silver nitrate followed by washing then immersion in an image developer containing formalin and sodium carbonate. There is no indication of color development with this stain procedure.
Poehling and Neuhoff, Electrophoresis 1981, 2, 141-147, describe a silver stain suitable for acrylamide gels of 0.5 to 1 mm thickness which requires a pretreatment with glutardialdehyde under controlled temperatures prior to staining with a diamine solution.
Marshall and Latner, Electrophoresis 1981, 2, 228-235, describe a silver stain method which requires a treatment with paraformaldehyde and sodium cacodylate prior to staining with a modified diamine solution wherein methylamine is substituted for ammonium hydroxide. We have described the basic staining procedure of the present invention in Electrophoresis 1981, 2 135-145 and said procedure was included in a comparative study of certain staining methods reported by Ochs, et al., Electrophoresis 1981, 2, 304-307.
With the exception of the 1980 Goldman, et al., procedure all of the silver stain techniques known heretofore only stain proteins in varying shades of brown or black.
The present invention provids a unique stain procedure which not only is highly sensitive, but also, enables one to stain a variety of substances including proteins in varying shades of color.